Analysis Of The Differential Expression Genes Of Gallbladder Cancer Cells After The Induction Of TGF-β1and The Role Of FGFBP1and WISP2in Gallbladder Cancer

Part I Analysis of the differential expression genes of gallbladder cancer cells before and after TGF-β1induced epithelial-mesenchymal transitionObjective:To analyze the differential expression genes between gallbladder cancer cells with TGF-β1induction and untreated gallbladder cancer cells by using DNA chips.Methods:The GBC-SD cells were divided into7groups. We chose0,0.5ng/ml,5ng/ml,10ng/ml as the concentrations of TGF-β1for induction of epithelial-mesenchymal transition (EMT), and the GBC-SD cells were induced for48or72hours. After the induction, the total RNA was extracted. Then the markers of epithelium (CDH1) and mesenchyme (VIM) were examed by RT-PCR. Finally, we demonstrated the group of5ng/ml TGF-β1and induced for72hours presented the most complete EMT. So this group was chosen for the further research and the total RNA was labeled with fluorescent probe. The hybridazition was performed by using the human genome oligonucleotide microarray containing21,522transcripts. The image data were initially collected by LuxScan10KA dual Channel laser scanner. Final analysis was conducted by MAS2.0.Results:The expressions of265genes were found changed significantly by the DNA chips, in which167genes were up-regulated and98were down-regulated. We further analyzed the function of those genes. According to the gene function annotation taxonomy classification (GO classification), they were involved in a couple of biological processes, such as cell cycle, mitosis, cell division, DNA repair, DNA replication and etc. Meanwhile, the pathway analysis was performed and these genes were related with the pathway of cell cycle, DNA polymerase, pyrimidine metabolism, ECM-receptor interaction, cell adhesion molecules (CAMs), p53signaling pathway and etc.Conclusion:According to the DNA chips,265genes that presented differential expression were identified. Among them,167genes were up-regulated and98were down-regulated. These genes were involved in a variety of cellular function and signaling pathways. Part II The significant expressions of FGFBP1and WISP2in gallbladder cancer and chronic cholecystitisObjective:To investigated the expressions of FGFBP1and WISP2in gallbladder cancer and chronic cholecystitis.Methods:The total RNA and protein were extracted from fresh tissue samples of15cases of gallbladder adenocarcinoma and15cases of chronic cholecystitis, respectively. Then RT-PCR and Western-blot were used to determine the expressions of FGFBP1and WISP2. Finally, statistic analysis was performed to help us to draw conclusions.Results:The expression of FGFBP1mRNA was significantly up-regulated in gallbladder adenocarcinoma than that in chronic cholecystitis (0.778±0.082vs0.467±0.081, P<0.01); the expression of WISP2mRNA was significantly down-regulated in gallbladder adenocarcinoma than that in chronic cholecystitis (0.202±0.046vs0.764±0.015, P<0.01). According to Western-blot analysis, the protein of FGFBP1increased apparently in gallbladder adenocarcinoma than that in chronic cholecystitis (0.851±0.069vs0.526±0.063, P<0.05); however, the protein of WISP2decreased significantly in gallbladder adenocarcinoma than that in chronic cholecystitis (0.454±0.056vs0.848±0.078,P<0.01)。Conclusion:The expression of FGFBP1was significantly higher in gallbladder adenocarcinoma than that in chronic cholecystitis; however, the expression of WISP2presented an opposite way. PartⅢ The influence of FGFBP1silencing by RNA interference on the biological characters of the GBC-SD cellsObjective:To determine the influence of FGFBP1silencing by RNA interference on the biological characters of the GBC-SD cells.Methods:The untreated GBC-SD cells and the GBC-SD cells transfected with unrelated sequence as controls. Liposome was used for the transfection of3vectors, FGFBP1-RNAi-1,2,3. The efficiency of transfection was examed by using fluorescence microscope. The most efficient vector was chosen for the following experiment. Then RT-PCR and Western-blot were performed for further confirmation of the transfection. MTT assay was used to detect the proliferation activity and the changes of invasive and migratory abilities were measured by transwell chamber invasion assay and scratch test.Results:The GBC-SD cells transfected with those vecors, showed green fluorescence by fluorescence microscope. The group of FGFBP1-RNAi-1exhibited the lightest green fluorescence. We considered FGFBP1-RNAi-1as the most efficient vector. Then RT-PCR and Western-blot were performed and the expression of FGFBP1was down-regulated even deleted as expected. Furthermore, MTT assay showed the proliferation of the GBC-SD cells in vitro was inhibited. The invasive and migratory abilities decreased significantly than the controls according to transwell chamber invasion assay and scratch test.Conclusion:The transfection of those vectors was successful. The FGFBP1silencing inhibited the proliferation, as well as the invasive and migratory abilities of the GBC-SD cells. PartIV Expressive levels of FGFBP1and WISP2and its clinicopathological significances in the benign and malignant lesions of the gallbladderObjective:To study on the expressive levels of FGFBP1and WISP2and detect their clinicopathological significances in the benign and malignant lesions of the gallbladder.Methods:En Vision immunohistochemical method for determining the expressions of FGFBP1and WISP2was used in routinely paraffin-embedded sections of surgical resected specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenoma (n=30), polyp (n=15), and chronic cholecystitis (n=35).Results:The positive rate of FGFBP1expression was significantly higher in gallbladder adenocarcinoma (57.4%) than that in peritumoral tissues (32.6%), adenoma (20.0%), adenomatous polyp (20.0%) and chronic cholecystitis (11.4%)(P<0.05or P<0.01); however, the positive rate of WISP2expression presented an opposite way. The negative cases of WISP2and/or positive ones of FGFBP1in the benign lesions showed moderately-or severely-atypical hyperplasia of gallbladder epitheli. The positive rates of WISP2were significanctly higher in the cases of well-differentiated adenocarcinoma, no-metastasis of lymph node, and no-invasiveness of regional tissues than those in the ones of poorly-differentiated adenocarcinoma, metastasis of lymph node, and invasiveness of regional tissues in gallbladder adenocarcinoma (P<0.05or P<0.01). The positive rates of FGFBP1were significanctly lower in the cases of well-differentiated adenocarcinoma, no-metastasis of lymph node, and no-invasiveness of regional tissues than those in the ones of poorly-differentiated adenocaarcinoma, metastasis of lymph node, and invasiveness of regional tissues in gallbladder adenocarcinoma (P<0.05or P<0.01). The high unconsistence was found between the expressive levels of FGFBP1and WISP2in gallbladder adenocarcinoma (χ2=16.47, P<0.01). Unitivariate Kaplan-Meier analysis showed that decreased expression of WISP2(P=0.13) or increased expression of FGFBP1(P=0.018) was associated with decreased overall survival. Multivariate Cox regression analysis showed that decreased expression of WISP2(P=0.012) and/or increased expression of FGFBP1(P=0.009) was an independent bad-prognostic predictor in gallbladder adenocarcinoma.Conclusions:The expression of FGFBP1and/or WISP2might be closely related to the carcinogenesis, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma.